Solution structure and peptide binding of the SH3 domain from human Fyn.

BACKGROUND: The Src family of tyrosine kinases is involved in the propagation of intracellular signals from many transmembrane receptors. Each member of the family contains two domains that regulate interactions with other molecules, one of which is the Src homology 3 (SH3) domain. Although structures have previously been determined for SH3 domains, and ideas about peptide-binding modes have been proposed, their physiological role is still unclear. RESULTS: We have determined the solution structure of the SH3 domain from the Src family tyrosine kinase Fyn in two forms: unbound and complexed with a peptide corresponding to a putative ligand sequence from phosphatidylinositol 3' kinase. Fyn SH3 shows the typical SH3 topology of two perpendicular three-stranded beta sheets and a single turn of 3(10) helix. The interaction of SH3 with three potential ligand peptides was investigated, demonstrating that they all bind to the same site on the molecule. A previous model for ligand binding to SH3 domains predicts binding in one of two orientations (class I or II), each characterized by a consensus sequence. The ligand with the closest match to the class I consensus sequence bound with highest affinity and in the predicted orientation. CONCLUSIONS: The Fyn SH3 domain has a well-defined structure in solution. The relative binding affinities of the three ligand peptides and their orientation within the Fyn SH3 complex were consistent with recently proposed models for the binding of 'consensus' polyproline sequences. Although the affinities of consensus and non-consensus peptides are different, the degree of difference is not very large, suggesting that SH3 domains bind to polyproline peptides in a promiscuous manner.

Localization and characterization of the hyaluronan-binding site on the link module from human TSG-6.

BACKGROUND: The interactions of hyaluronan (HA) with proteins are important in extracellular matrix integrity and leukocyte migration and are usually mediated by a domain termed a Link module. Although the tertiary structure of a Link module has been determined, the molecular basis of HA-protein interactions remains poorly understood. RESULTS: Isothermal titration calorimetry was used to characterize the interaction of the Link module from human TSG-6 (Link_TSG6) with HA oligosaccharides of defined length (HA(4)-HA(16)). All oligomers bound (except HA(4)) with K(d) values ranging from 0.2-0.5 microM at 25 degrees C. The reaction is exothermic with a favourable entropy and the thermodynamic profile is similar to those of other glycosaminoglycan-protein interactions. The HA(8) recognition site on Link_TSG6 was localized by comparing nuclear magnetic resonance (NMR) spectra from a 1:1 complex with free protein. Residues perturbed on HA binding include both amino acids that are likely to be directly involved in the interaction (i.e., Lys11, Tyr59, Asn67, Phe70, Lys72 and Tyr78) and those affected by a ligand-induced conformational change in the beta4/beta5 loop. The sidechain of Asn67 becomes more rigid in the complex suggesting that it is in close proximity to the binding site. CONCLUSIONS: In TSG-6 a single Link module is sufficient for a high-affinity interaction with HA. The HA-binding surface on Link_TSG6 is found in a similar position to that suggested previously for CD44, indicating that its location might be conserved across the Link module superfamily. Here we find no evidence for the involvement of linear sequence motifs in HA binding.

TSG-6 interacts with hyaluronan and aggrecan in a pH-dependent manner via a common functional element: implications for its regulation in inflamed cartilage.

Cartilage matrix is stabilised by the interactions of proteins with hyaluronan (HA). We compare the pH dependences of HA binding by aggrecan, link protein and TSG-6. Aggrecan and link protein exhibit maximal binding across a wide pH range (6.0-8.0). TSG-6, a protein that is only produced during inflammation, binds maximally at about pH 6.0 but shows a dramatic loss of function with increasing pH. TSG-6 also interacts with aggrecan, with a similar pH dependence, and this can be inhibited by HA. Thus, a common binding surface on TSG-6 may be involved in HA and aggrecan binding. We propose that TSG-6 is involved in matrix dissociation and that this is regulated by pH gradients in cartilage.

Method for quantitative refolding of the link module from human TSG-6.

We have developed a procedure for the quantitative refolding of the Link module from human tumor necrosis factor-stimulated gene 6. This significantly simplifies the previously described method of production of this protein domain (Day et al., Protein Expression Purif. 8, 1-16, 1996). The refolding is carried out under nondenaturing conditions at pH 6.0 in the presence of a 100-fold molar excess of beta-mercaptoethanol. After 2 days the starting material, which consists of three species that differ only with respect to their disulfide bond organization, has rearranged to give a single homogeneous species with the correct disulfide bridges. This method allows the production of about 20 mg of folded protein per liter of Escherichia coli culture.

Synthesis of pseudopeptides with sulfoximines as chiral backbone modifying elements.

The synthesis of pseudopeptides with a chiral alpha-sulfonimidoylcarboxy moiety in the backbone is described. Starting from readily available (Ss)-S-methyl S-phenyl sulfoximine and various cyclic and acyclic alpha-amino acids the desired products are obtained in good yields with peptide coupling methodology. Specific secondary structures caused by intramolecular hydrogen bonds may be adopted. Results of NMR studies to reveal conformational preferences will be discussed.

Characterization of a functional hyaluronan-binding domain from the human CD44 molecule expressed in Escherichia coli.

The CD44 molecule is a widely distributed cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan. The ligand-binding site which is located in the membrane distal portion of the molecule encompasses a region of approximately 100 amino acids termed the Link domain, a structural unit that is conserved among members of the Hyaladherin superfamily which includes cartilage link protein, aggrecan, and tumor necrosis factor-stimulated gene-6 (TSG-6). In contrast to these other Hyaladherins, however, the ligand-binding domain of CD44 appears to extend beyond the Link domain to involve additional basic residues located toward the membrane proximal region. Furthermore, recent molecular modeling studies indicate that within the CD44 Link domain itself, the spatial arrangement of critical residues involved in HA binding is likely to differ significantly from the prototypic TSG-6 Link module. In order to obtain material to solve the CD44 solution structure we have developed an optimized method for the expression and purification of functionally active CD44 ectodomains encompassing both the Link module and the additional downstream HA-binding residues in Escherichia coli. Here we describe the details of the method which involves solubilization of recombinant CD44 from inclusion bodies in 8 M urea, followed by refolding and purification of intact monomers using size-exclusion and reverse-phase chromatography. We show the method yields CD44 molecules that (1) retain reactivity with a panel of conformation-sensitive antibodies, (2) possess similar hyaluronan-binding characteristics to authentically folded CD44 molecules expressed in eukaryotic cells, and (3) display one-dimensional NMR spectra that indicate the presence of a single conformational species. This method should enable sufficient amounts of functional CD44 Link module to be produced for comprehensive structural analyses by multidimensional NMR spectroscopy.